Syntheses and evaluation of macrocyclic engelhardione analogs as antitubercular and antibacterial agents

DOI: 10.1038/ja.2013.21

Source and publish data:

Journal of Antibiotics p. 319 - 325 (2013)

Update date:2022-08-03

Topics:

Authors:

Shen, Li

M Maddox, Marcus

Adhikari, Sudip

Bruhn, David F.

Kumar, Manish

Lee, Robin E.

Hurdle, Julian G.

Lee, Richard E.

Sun, Dianqing

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Article abstract of DOI:10.1038/ja.2013.21

The natural product engelhardione is an underexplored chemotype for developing novel treatments for bacterial infections; we therefore explored this natural product scaffold for chemical diversification and structure-activity relationship studies. Macrocy

Full text of DOI:10.1038/ja.2013.21

The Journal of Antibiotics (2013) 66, 319–325

www.nature.com/ja

ORIGINAL ARTICLE

Syntheses and evaluation of macrocyclic

engelhardione analogs as antitubercular and

antibacterial agents

Li Shen¹, Marcus M Maddox², Sudip Adhikari³, David F Bruhn², Manish Kumar³, Robin E Lee²,

Julian G Hurdle³, Richard E Lee²and Dianqing Sun¹

The natural product engelhardione is an underexplored chemotype for developing novel treatments for bacterial infections;

we therefore explored this natural product scaffold for chemical diversiﬁcation and structure–activity relationship studies.

Macrocyclic engelhardione and structural regioisomers were synthesized using a series of aldol condensations and selective

hydrogenations to generate the 1,7-diarylheptan-3-one derivatives, followed by microwave-assisted intramolecular Ullmann

coupling to afford a series of macrocyclic diaryl ether analogs. An extended macrocyclic chemical library was then produced

by oxime formation, reductive amination and O-alkylation. Antibacterial evaluation revealed that the reductive amination

derivatives 7b and 7d showed moderate activities (minimum inhibitory concentrations: 12.5–25 mg ml^ꢀ1) against

Mycobacterium tuberculosis and Gram-positive pathogens, as well as anti-Gram-negative activity against an efﬂux impaired

Escherichia coli strain. These results provide validated leads for further optimization and development.

The Journal of Antibiotics (2013) 66, 319–325; doi:10.1038/ja.2013.21; published online 3 April 2013

Keywords: antibacterial; antitubercular; diarylheptanoid; engelhardione; macrocycle; pterocarine

INTRODUCTION

reported by the Nagai’s group in 1976,⁸diverse diarylheptanoids^9–16

Because of the emergence and spread of drug-resistant Mycobacterium have been isolated and found to mediate a variety of biological

tuberculosis and other pathogenic bacterial infections, there is an activities (Figure 1). One such example, engelhardione, was recently

urgent need to discover new chemotype antitubercular and antibac- isolated from the roots of Engelhardia roxburghiana and reported to

terial agents with novel mechanisms of action.^1,2Only ﬁve novel show potent antituberculosis activity with a minimum inhibitory

chemical classes of antibiotics, exempliﬁed by linezolid, daptomycin, concentration (MIC) of 0.2 mg ml^{ꢀ1 17}

retapamulin, ﬁdaxomicin and bedaquiline, have been introduced As our continued effort to develop natural products-derived novel

into the clinic since the early 1960s.³Among antibacterial antibacterial agents, we have been interested in the chemical

discovery strategies, whole-cell-based phenotypic screens of small- modiﬁcation of emerging natural product scaffolds. Inspired by

molecule and/or natural product-like libraries, followed by target the reported potent antitubercular activity of engelhardione and the

deconvolution and identiﬁcation, remain an attractive and efﬁcient limited attention given to exploring this macrocyclic molecule,

approach.⁴

we directed medicinal chemistry efforts toward this promising natural

Natural products represent one of the most valuable sources for product scaffold. Consequently, we recently reported the ﬁrst total

novel bioactive molecules and chemical diversity in drug discovery.⁵synthesis of the published structure (1a) of engelhardione and

Indeed, most antibiotics in clinical use are natural products, this effort led to its structural revision to that of pterocarine

semisynthetic and/or natural product-inspired derivatives.⁶Notably, (1b; Figure 1).¹⁸The structural revision was also subsequently

most clinically used natural product antibiotics are derived from conﬁrmed by the Natarajan’s group⁹and the Chen’s group.¹⁹To

microorganisms such as bacteria and fungi, and no plant-derived further improve the efﬁciency of macrocyclization, we developed an

antibacterial agents have been used clinically.³Macrocyclic efﬁcient and modular microwave-assisted macrocyclization via

diarylheptanoids belong to a chemical class of bioactive naturally intramolecular Ullmann coupling and investigated the scope and

occurring phytochemicals, which display a characteristic diphenyl generality of a panel of substrates with different linkers, ring sizes and

ether motif linked by a seven carbon bridge.⁷As acerogenin A, the substitution patterns.²⁰To extend the medicinal chemistry effort of

ﬁrst member in the cyclic diarylheptanoid class, was isolated and this work and to investigate if this cyclic diarylheptanoid architecture

¹Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawai’i at Hilo, Hilo, HI, USA; ²Department of Chemical Biology and Therapeutics, St Jude

Children’s Research Hospital, Memphis, TN, USA and ³Department of Biology, University of Texas at Arlington, Arlington, TX, USA

Correspondence: Dr D Sun, Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawai’i at Hilo, Suite 300, Hilo, HI 96720, USA.

E-mail: dianqing@hawaii.edu

Received 10 December 2012; revised 21 February 2013; accepted 26 February 2013; published online 3 April 2013

Novel diarylheptanoid analogs as antibacterial agents

L Shen et al

320

OH OH

acerogenin B: R = OH

acerogenin L: R = O

MeO

acerogenin A: R = OH

acerogenin C: R = O

aceroside B₁

acerogenin glycoside

MeO

R₂O

MeO

engelhardione/pterocarine

(1b): R₂= H

galeon: R₂= Me

the published structure

of engelhardione

MeO

myricatomentogenin: R = H

juglanin A: R = Me

ovalifoliolatin A: R = OH

ovalifoliolatin B: R = H

platycarynol

reported MIC: 0.2 µg/mL

against M. tuberculosis (H37Rv)

Figure 1 Chemical structures of bioactive diarylheptanoids with a cyclic diphenyl ether moiety. A full color version of this ﬁgure is available at The Journal

of Antibiotics journal online.

R¹

R⁴

R¹

R⁴

R¹

R²

R¹

R²

R³

R²

R³

3a-c

2a: R¹= OMe; R²= OH; R³= Br; R⁴= OMe;

2b: R¹= OH; R²= OMe; R³= Br; R⁴= OMe;

2c: R¹= OMe; R²= OH; R³= OMe; R⁴= Br;

R⁴

R³

4a R¹= R⁴= OMe

4b R²= R⁴= OMe

4c R¹= R³= OMe

1a R¹= R⁴= OH

1b R²= R⁴= OH

1c R¹= R³= OH

Scheme 1 Synthesis of engelhardione (1a) and its regioisomers (1b and c). Reagents and conditions: (a) 5–10% Pd/C, H₂, Ph₂S (0.05 equiv.), CHCl₃,

7–18 h, 78–90%; (b) CuO, K₂CO₃, pyridine, 1751C, 4.5–5 h, 52–58%; and (c) AlCl₃, CH₂Cl₂, reﬂux, 11–25h, 31–62%.

possesses any tractable antibacterial activity, herein we report the quantitative yield (Scheme 2). Second, reaction of 4a with different

synthesis, antibacterial evaluation and preliminary structure–activity substituted hydroxylamine hydrochloride in the presence of pyridine

relationships of this class of macrocyclic diarylheptanoids against in ethanol at room temperature afforded the oxime derivatives 6a–c.²¹

M. tuberculosis and

broad panel of Gram-positive and In the cases of 6b and 6c, based on their ¹H NMR spectra, a mixture

Gram-negative pathogens. This work constitutes the ﬁrst systematic of E and Z oxime isomers in an approximate ratio of 1:1 and 1:2 was

report describing the antitubercular and antibacterial evaluation of obtained, respectively. Initial attempts to prepare Schiff base imines

synthetic engelhardione, pterocarine and related structural analogs. from the reaction of 4a with amines were unsuccessful because of the

Our preliminary mechanistic study identiﬁed that lead compounds facile decomposition of imine products during the work-up and

inhibited several key macromolecular processes (DNA, RNA and puriﬁcation. The one-pot syntheses of the secondary amine analogs

protein).

7a–e were achieved by reacting 4a with an array of different

substituted amines, followed by NaBH₄-mediated reduction in a

sealed tube under solvent-free conditions. In all cases, the reactions

proceeded smoothly to give products 7a–e in fair to good yields

RESULTS AND DISCUSSION

Chemistry

As illustrated in Scheme 1, starting from 1,7-diarylhepten-3-one (47–78%).

2a–c,¹⁸the proposed structure (1a) of engelhardione, pterocarine (1b)

Next, synthesis of different substituted engelhardione O-alkyl

and their regioisomer (1c) were synthesized by a series of cross analogs 13a–f was carried out by employing an orthogonal protecting

aldol condensations and selective hydrogenations, affording linear strategy and adapting the similar synthetic scheme as used for 4a

1,7-diaryl-3-ketones as a key intermediate (3a–c), followed by (Scheme 3). The conjugated diarylhepten-3-one 2d was obtained by

intramolecular Ullmann reactions to give the macrocyclic Claisen–Schmidt condensation of 8 with methoxymethyl ether

architectures 4a–c and ﬁnal O-demethylation (Scheme 1).

(MOM)-protected benzaldehyde 10,²²followed by chemoselective

To evaluate the inﬂuence of the carbonyl group on the antibacterial hydrogenation to give the 1,7-diphenylheptan-3-one 3d.²³

activity, compounds 5–7 were then designed and synthesized. First, Intramolecular macrocyclic Ullmann reaction was then performed

the carbonyl group of engelhardione dimethyl ether (4a) was reduced under microwave irradiation to give the MOM-protected macrocycle

to the racemic secondary alcohol (5) by reaction with NaBH₄in a 11 in good yield (85%). The MOM-deprotected macrocyclic phenol

The Journal of Antibiotics

Novel diarylheptanoid analogs as antibacterial agents

L Shen et al

321

R²

MeO

R¹

OR¹

MeO

Scheme 2 Synthesis of engelhardione derivatives 5–7. Reagents and conditions: (a) NaBH₄, MeOH, 0 1C to room temperature (rt), 20 min, 100%; (b)

hydroxylamine hydrochloride, NaOH, MeOH, reﬂux, 6 h or R¹ONH₂ꢁ HCl, pyridine, EtOH, rt, 78–91%; and (c) (i) R²NH₂, solvent-free, 701C, overnight; (ii)

NaBH₄, 70 1C, 10min, 47–78%.

OHC

MeO

OMOM

MeO

OMOM

MeO

R = H

10 R = MOM

R =

13d

13a

13b

13e

13f

MeO

MOMO

13c

Scheme 3 Synthesis of engelhardione O-alkylated derivatives 13a–f. Reagents and conditions: (a) methyl chloromethyl ether (MOMCl), DIPEA, CH₂Cl₂, 0 1C

to room temperature (rt), overnight, 94%; (b) 10% NaOH, EtOH, rt, overnight, 62%; (c) 10% Pd/C, H₂, Ph₂S (0.05 equiv.), CHCl₃, 18 h, 78%; (d) CuO,

K₂CO₃, pyridine, MW, 220 1C, 85%; (e) conc. HCl, MeOH, rt, overnight, 94%; and (f) for 13a–d: RBr, K₂CO₃, acetone, reﬂux, 47–94%; for 13e: DIPEA,

CH₃CN, reﬂux, overnight, 90%; for 13f: NaH, DMF, 01C to rt, 2 h, 58%.

12 was then prepared by deprotection of 11 with concentrated HCl in corniculatolides, close structural lactone analogs of engelhardione/

methanol at room temperature in high yield. Finally, in the presence pterocarine diarylheptanoids, did not inhibit the growth of

of appropriate bases, 12 was subjected to the reactions with different M. tuberculosis at 100 mg ml^ꢀ1

alkyl bromides to afford a series of O-alkylated macrocycles 13a–f in

47–94% yields.

The oxime derivative 6a showed slight activity against S. aureus

(ATCC 29213) (MSSA) (MIC¼ 100 mg ml^ꢀ1). Improved activities

were obtained for the reductive amination products 7b–e; in

particular, compounds 7b and 7d containing secondary amine

Biology

To evaluate the potential for antituberculosis and broad-spectrum functionality exhibited antibacterial activities against M. tuberculosis,

antibacterial activities, all the linear 1,7-diarylheptanoid intermediates E. faecalis, S. aureus MSSA and MRSA with MIC values of

and macrocyclic engelhardione analogs were tested against 12.5–25 mg ml^ꢀ1

. Interestingly, compounds 7b–e also showed

M. tuberculosis and a panel of Gram-positive and Gram-negative moderate activity against an E. coli strain that is deﬁcient in TolC, a

pathogens including Enterococcus faecalis, Staphylococcus aureus critical component of the AcrAB–TolC efﬂux system that pumps

(MSSA), Staphylococcus aureus (MRSA), Escherichia coli (K12), antibiotics out of the periplasm.²⁵In contrast, compound 7a with an

Escherichia coli (DTolC), Klebsiella pneumonia and Pseudomonas additional morpholine motif in this N-substituted amine series was

aeruginosa. The results are shown in Table 1 and preliminary not active, possibly due to the increased polarity and decreased

structure–activity relationships are summarized below.

cellular membrane penetration. It is worthwhile to note that

Most linear chain diarylheptanoid compounds 2 and 3 were compounds 7b–e were not active toward wild-type E. coli strain.

inactive. The engelhardione analogs 1a–c and their corresponding This result suggests that these compounds may be subject to efﬂux

methyl ether derivatives 4a–c only showed marginal antibacterial pump-mediated resistance mechanism and explains their lack of

activities. Unfortunately, both the originally published structure¹⁷activity against Gram-negative bacteria.

(1a) of engelhardione and pterocarine (1b) only showed weak

The O-alkylated cyclic derivative 13e with an isopropyl group

antituberculosis activity (200mg ml^ꢀ1) in our assay. These results showed moderate antituberculosis activity (MIC¼ 25mg ml^ꢀ1), but

are consistent with a recent report by Reddy and co-workers²⁴that all the other compounds in this series were inactive.

The Journal of Antibiotics

Novel diarylheptanoid analogs as antibacterial agents

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322

Table 1 Antitubercular and antibacterial activities (lg ml^ꢀ1) of linear diarylheptanoid intermediates and macrocyclic engelhardione analogs

M. tuberculosis

E. faecalis (ATCC

S. aureus (ATCC

S. aureus (NRS 70) E. coli

E. coli

K. pneumonia

P. aeruginosa

Compound

(H37Rv)

33186)

29213) (MSSA)

(MRSA)

(K12)

(TolC)

(ATCC 33495)

(PAO1)

4200

200

4200

200

4200

200

4200

200

4200

1b (engelhardione/

200

pterocarine)

200

4200

200

4200

100

4200

4200 4200

4200

200

12.5

100

12.5

100

4200

200

4200

0.78

4200

0.39

4200

200

13a

13b

13c

13d

13e

13f

Vancomycin

4200

o0.2

4200 4200

4200

0.032–0.064 (INH

as control)

100

Abbreviations: INH, isoniazid; ND, not determined.

120

100

120

100

120

100

Protein

DNA

RNA

Figure 2 Inhibition of macromolecular synthesis by 7b–d. Indicated are the standard error of means from three or more biologically independent

experiments. SM, streptomycin (MIC, 1.6 mg ml^ꢀ1); CPFX, ciproﬂoxacin (MIC, 0.40 mg ml^ꢀ1); RFP, rifampicin (MIC, 12.5 mg ml^ꢀ1).

The Journal of Antibiotics

Novel diarylheptanoid analogs as antibacterial agents

L Shen et al

323

To explore the basis for the antibacterial activities of selected (2H, m), 1.56ꢀ1.49 (2H, m) and 1.19 ꢀ1.11 (2H, m) p.p.m. ¹³C NMR (CDCl₃,

100 MHz): d ¼ 158.6, 150.3, 147.7, 147.6, 145.4, 134.9, 133.2, 125.6, 122.7,

compounds, their effects on the biosynthesis of key macromolecules

were examined against the E. coli TolC deletion mutant. These effects

were examined at low and elevated concentrations (that is, 1 and

8 ꢂ the MIC) to determine concentration-dependent effects that may

121.2, 118.0, 114.0, 113.9, 56.3, 56.2, 35.5, 31.9, 30.9, 29.5, 28.0 and 22.2 p.p.m.

ESI-MS: calc. for C₂₁H₂₆NO₄[M þ H]^þ: 356.2; found: 356.3. HPLC purity:

98.4% (254 nm), t_R: 6.85min; 97.7% (220 nm), t_R: 6.86 min.

Compound 6b: 6b was prepared as described above for compound 6a from

point to a speciﬁc process as the main antibacterial target.

Interestingly, the biosynthesis of DNA, RNA and protein were all

4a (20 mg, 0.059mmol) with methoxylamine hydrochloride (19.7mg,

0.24mmol). After ethanol was removed, the solid was dissolved in dichloro-

highly inhibited by the compounds 7b–d at 1 and 8 ꢂ their MICs

methane and washed with water. The organic layer was dried over anhydrous

Na₂SO₄and then ﬁltered. Solvent was removed by evaporation to give the

crude product, which was further puriﬁed by ﬂash column chromatography on

(Figure 2), which may be indicative of a nonspeciﬁc mode of

action, possibly at the cell envelope. This is similar to the effects

observed in cells treated with the macrocycles daptomycin and silica gel (hexanes/ethyl acetate, 88:12) to give 18.2 mg white powder as a

mixture of Z- and E-oxime isomers (approximately 1:1 ratio based on ¹H

telavancin that target the bacterial envelope and disrupt multiple

processes in cells.^26,27

In summary, a small focused library of diarylheptanoid engelhar-

NMR spectrum) (84%). ¹H NMR (CDCl₃, 400MHz): d ¼ 6.92ꢀ6.86 (3H, m),

6.76ꢀ6.71 (2H, m), 6.43 (0.5H, s), 6.30 (0.5H, s), 3.92 (1.5H, s), 3.90

(1.5H, s), 3.88 (1.5H, s), 3.86 (1.5H, s), 3.81 (1.5H, s), 3.71 (1.5H, s),

2.83ꢀ2.80 (2H, m), 2.52ꢀ2.49 (2H, m), 2.45ꢀ2.39 (2H, m), 2.08 (1H, t,

functionalities were designed and synthesized. Antituberculosis

dione and derivatives bearing the oxime, and N- and O-alkylated

J ¼ 7.4 Hz), 1.80 ꢀ1.76 (1H, m), 1.56ꢀ1.53 (1H, m), 1.48ꢀ1.41 (1H, m),

and antibacterial evaluation revealed that the N-substituted

1.25ꢀ1.18 (1H, m) and 1.17ꢀ1.09 (1H, m)p.p.m. ¹³C NMR (CDCl₃,

secondary amine derivatives demonstrated moderate antitubercular

and antibacterial activities. In particular, compound 7b with a

phenethylamine substituent and compound 7d with a n-hexylamino

100 MHz): d ¼ 161.1, 158.7, 150.4, 149.4, 148.4, 148.0, 147.7, 146.6, 146.0,

134.3, 134.0, 133.6, 133.1, 125.4, 125.2, 123.3, 122.3, 121.2, 118.9, 118.6, 118.2,

112.9, 112.8, 112.5, 112.4, 61.2, 61.0, 56.1, 35.4, 33.0, 32.2, 32.0, 31.5, 30.2,

moiety demonstrated the most potent antibacterial activity against 29.2, 28.9, 28.2, 28.0, 23.7 and 22.4p.p.m. ESI-MS: calc. for C₂₂H₂₈NO₄

[Mþ H]^þ: 370.2; found: 370.3. HPLC purity: 98.4% (254nm), t_R: 7.51min;

M. tuberculosis, E. faecalis and S. aureus with MICs ranging from 12.5

to 25 mg ml^ꢀ1. Based on the existing structure–activity relationships,

99.4% (220 nm), t_R: 7.50 min.

Compound 6c: 6c was prepared as described above for compound 6b from

4a (18 mg, 0.053 mmol) with benzyloxylamine hydrochloride (33.9 mg,

further synthesis and optimization are ongoing in an effort to obtain

compounds with improved antibacterial potency and therapeutic

potential.

0.212 mmol). The reaction mixture was puriﬁed by ﬂash column chromato-

graphy on silica gel (hexanes/ethyl acetate, 90:10) to give 21.4mg white powder

as a mixture of Z- and E-oxime isomers (approximately 1:2 ratio based on ¹H

EXPERIMENTAL PROCEDURE

Chemistry

All reagents and solvents obtained from commercial sources were used without

further puriﬁcation. Reactions were monitored by high-performance liquid

NMR spectrum) (91%). ¹H NMR (CDCl₃, 400MHz): d ¼ 7.36ꢀ7.29 (3H, m),

7.26ꢀ7.22 (3H, m), 7.12ꢀ7.10 (2H, m), 6.92 ꢀ6.88 (3H, m), 6.83 (1H, d,

J ¼ 8.2 Hz), 6.79 (0.5H, d, J ¼ 8.4 Hz), 6.75 (0.5H, d, J ¼ 8.2 Hz), 6.70 (1H, d,

J ¼ 8.0 Hz), 6.63ꢀ6.15 (1.5H, m), 6.28 (1.5H, s), 5.05 (1H, s), 4.94 (1H, s),

chromatography (HPLC) with a Shimadzu LC-20A series HPLC system

(Shimadzu Scientiﬁc Instruments, Carlsbad, CA, USA). Hydrogenation reactions

3.93 (1.5H, s), 3.92 (3H, s), 3.88 (3H, s), 3.85 (1.5H, s), 2.85ꢀ2.79 (3H, m),

2.52ꢀ2.49 (1H, m), 2.47ꢀ2.44 (4H, m), 2.42 ꢀ2.39 (1H, m), 2.36 ꢀ2.33 (4H,

were carried out using domnick hunter NITROX UHP-60H hydrogen generator

(Parker Hanniﬁn Corp, Cleveland, OH, USA). Reactions in sealed tube were

carried out using Q-Tube pressure tube reactors from Q Lab tech (Waterford,

m), 2.15 (2H, t, J ¼ 6.8Hz), 1.80ꢀ1.76 (1H, m), 1.58ꢀ1.52 (1H, m),

1.25ꢀ1.22 (3H, m) and 1.09ꢀ1.04 (2H, m)p.p.m. ¹³C NMR (CDCl₃,

100 MHz): d ¼ 161.4, 158.6, 150.4, 148.9, 148.7, 147.1, 146.9, 138.5, 138.3,

CT, USA). Microwave-assisted synthesis was performed by Biotage Initiator 8

Exp Microwave System (Biotage, LLC, Charlotte, NC, USA). Flash column

chromatography was performed using a Biotage Isolera One system and a

Biotage SNAP cartridge (Biotage). ¹H NMR and ¹³C NMR and 2D HSQC,

COSY, HMBC spectra were recorded employing a Bruker AM-400 spectrometer

(Bruker BioSpin Corporation, Fremont, CA, USA). Chemical shifts were

expressed in p.p.m. and J values were in Hz. Mass spectra were recorded on a

Varian 500-MS IT mass spectrometer using ESI (Agilent Technologies, Santa

Clara, CA, USA). The purity of compounds was determined by analytical HPLC

134.2, 134.0, 133.8, 133.1, 128.6, 128.3, 128.2, 128.1, 127.7, 127.6, 125.2, 124.9,

123.7, 122.3, 121.4, 118.8, 118.1, 117.3, 112.8, 112.7, 112.4, 112.2, 75.6, 75.5,

56.1 (2C), 35.5, 33.2, 32.0, 31.9, 31.3, 29.6, 29.1, 28.8, 28.3, 28.0, 24.1 and

22.3p.p.m. ESI-MS: calc. for C₂₈H₃₂NO₄[Mþ H]^þ: 446.2; found: 446.3.

HPLC purity: 98.5% (254 nm), t_R: 7.93 min; 98.9% (220nm), t_R: 7.94min.

Compound 7a: A mixture of engelhardione dimethyl ether 4a (15.0 mg,

0.044 mmol) and 2-morpholinoethanamine (60 ml, 0.46 mmol) in a sealed tube

was heated at 701C for overnight. NaBH₄(16.7mg, 0.44 mmol) was then

added, and the mixture was stirred at 701C for 10min. The reaction mixture

was cooled to room temperature, and water (2ml) was added carefully, and the

resulting mixture was extracted with ethyl acetate (2ꢂ 10ml). The organic

layers were combined, washed with brine and dried over anhydrous Na₂SO₄.

The solvent was removed by evaporation to give the crude product, which was

further puriﬁed by ﬂash column chromatography on silica gel (DCM/MeOH,

91:9) to give 12mg of white powder (60%). ¹H NMR (CDCl₃, 400 MHz):

d ¼ 6.89 ꢀ6.85 (3H, m), 6.78 (1H, dd, J ¼ 8.2 and 1.4 Hz), 6.62 (1H, s), 6.58

(1H, d, J ¼ 1.7 Hz), 3.89 (6H, 2s), 3.73 (4H, t, J ¼ 4.6 Hz), 2.88 ꢀ2.70 (4H, m),

2.66ꢀ2.56 (3H, m), 2.48ꢀ2.44 (4H, m), 2.42 ꢀ2.30 (2H, m), 1.87 ꢀ1.80 (1H,

m), 1.72 ꢀ1.61 (1H, m), 1.59ꢀ1.38 (4H, m), 1.23ꢀ1.15 (1H, m) and

0.94ꢀ0.85 (1H, m)p.p.m. ¹³C NMR (CDCl₃, 100 MHz): d ¼ 149.5, 149.0,

147.7, 147.2, 133.6, 132.3, 124.4, 124.3, 118.2, 118.0, 112.6 (2C), 67.0, 56.5,

56.1 (2C), 54.5, 53.5, 42.1, 33.1, 32.9, 32.2, 29.8, 28.1 and 23.4p.p.m. ESI-MS:

calc. for C₂₇H₃₉N₂O₄[Mþ H]^þ: 455.3; found: 455.4. HPLC purity: 98.5%

(254nm), t_R: 5.36 min; 98.8% (220 nm), t_R: 5.36 min.

using a Gemini, 3 mm, C18, 110 A column (50 mm ꢂ 4.6 mm; Phenomenex,

Torrance, CA, USA) and a ﬂow rate of 1.0 ml min^ꢀ1. Gradient conditions used

were: solvent A (0.1% triﬂuoroacetic acid in water) and solvent B (acetonitrile):

0–2.00 min, 100% A, 2.00–7.00 min, 0–100% B (linear gradient), 7.00–8.00 min,

100% B, and UV detection at 254 and 220 nm.

Syntheses of engelhardione (1a), pterocarine (1b), regioisomer (1c) and

their corresponding synthetic precursors (2–4) were described previously.^18,20

Syntheses of 2d, 3d, 5 and 10–12 were described in the Supplementary

Information.

Compound 6a: To a suspension of hydroxylamine hydrochloride (13.6mg,

0.196mmol) in ethanol (2 ml), 15.8ml (0.196mmol) of pyridine was added

dropwise, followed by the addition of engelhardione dimethyl ether 4a

(16.5 mg, 0.049mmol). The reaction was stirred at room temperature for 7h.

The ethanol was evaporated to give a white powder, which was washed with

water and ethyl acetate to give the product as a white powder (91%). ¹H NMR

(DMSO-d₆, 400MHz): d ¼ 10.34 (1H, s), 7.10 (1H, d, J ¼ 8.3 Hz), 7.05 (1H, d,

Compound 7b: 7b was prepared as described above for compound 7a from

J¼ 8.3 Hz), 6.97 (1H, dd, J ¼ 8.3 and 2.1 Hz), 6.82 (1H, dd, J ¼ 8.3 and 2.0 Hz), 4a (18.2 mg, 0.054mmol) with 2-phenylethanamine to give 12mg of product

6.61 (1H, d, J¼ 2.1 Hz), 6.23 (1H, d, J¼ 2.0Hz), 3.86 (3H, s), 3.81 (3H, s), (50%). ¹H NMR (CDCl₃, 400 MHz): d ¼ 7.36 ꢀ7.32 (2H, m), 7.29ꢀ7.21

2.85 ꢀ2.82 (2H, m), 2.51 ꢀ2.48 (2H, m), 2.40ꢀ2.37 (2H, m), 1.77 ꢀ1.72 (3H, m), 6.86 (1H, d, J ¼ 8.2Hz), 6.79 (1H, d, J ¼ 8.2 Hz), 6.75 (1H, dd,

The Journal of Antibiotics

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J ¼ 8.3 and 1.2 Hz), 6.63 (1H, d, J ¼ 1.4 Hz), 6.54 (1H, d, J ¼ 1.0 Hz), 6.44 (1H,

(47%). ¹H NMR (CDCl₃, 400 MHz): d ¼ 6.91 (1H, d, J ¼ 8.4 Hz), 6.88 ꢀ6.85

d, J ¼ 8.1 Hz), 3.89 (3H, s), 3.88 (3H, s), 2.99ꢀ2.90 (1H, m), 2.85ꢀ2.70 (2H, m), 6.76 (1H, dd, J ¼ 8.2 and 1.8 Hz), 6.51 (1H, d, J ¼ 1.8 Hz), 6.37

(3H, m), 2.66ꢀ2.61 (1H, m), 2.58ꢀ2.52 (1H, m), 2.41ꢀ2.32 (2H, m), 2.02 (1H, d, J ¼ 1.8 Hz), 5.48 (1H, t, J ¼ 6.0 Hz), 5.07 (1H, t, J ¼ 6.2 Hz), 4.63

(1H, t, J ¼ 9.6 Hz), 1.75 ꢀ1.68 (1H, m), 1.58ꢀ1.37 (4H, m), 1.24ꢀ1.09 (2H,

(2H, d, J ¼ 6.3 Hz), 3.89 (3H, s), 2.86 ꢀ2.83 (2H, m), 2.61ꢀ2.58 (2H, m),

m) and 0.91ꢀ0.81 (1H, m)p.p.m. ¹³C NMR (CDCl₃, 100MHz): d ¼ 149.4, 2.51 ꢀ2.48 (2H, m), 2.20 (2H, t, J ¼ 7.5 Hz), 2.12ꢀ2.01 (4H, m), 1.69 ꢀ1.59

148.7, 147.7, 147.1, 140.0, 133.7, 133.2, 128.8, 128.5, 126.3, 124.5, 124.0, 118.9, (9H, m), 1.57 ꢀ1.52 (2H, m) and 1.34 ꢀ1.26 (2H, m)p.p.m. ¹³C NMR

117.4, 112.4 (2C), 56.1 (2C), 53.9, 48.1, 36.3, 33.9, 33.5, 33.1, 29.9, 27.9 and (CDCl₃, 100 MHz): d ¼ 211.0, 148.9, 148.6, 147.4, 147.2, 140.1, 134.0, 133.6,

23.5p.p.m. ESI-MS: calc. for C₂₉H₃₅NO₃[Mþ H]^þ: 446.3; found: 446.4. 131.7, 124.5, 123.9, 122.9, 120.3, 119.0, 118.9, 115.8, 113.0, 66.6, 56.3, 43.7,

HPLC purity: 96.5% (254 nm), t_R: 6.06 min; 98.5% (220nm), t_R: 6.06min.

Compound 7c: 7c was prepared as described above for compound 7a from

41.6, 39.5, 33.1, 29.2, 27.8, 26.3, 25.7, 21.3, 17.7 and 16.6p.p.m. ESI-MS: calc.

for C₃₀H₃₉O₄[Mþ H]^þ: 463.3; found: 463.4. HPLC purity: 98.7% (254nm),

4a (15.0 mg, 0.044 mmol) with benzylamine to give 9 mg of product (47%). ¹H t_R: 8.13 min; 99.4% (220nm), t_R: 8.13min.

NMR (CDCl₃, 400 MHz): d ¼ 7.32ꢀ7.24 (5H, m), 6.87 (1H, d, J ¼ 8.2 Hz),

Compound 13c: 13c was prepared as described above for compound 13a

6.83 (1H, d, J ¼ 8.2Hz), 6.75 (1H, dd, J ¼ 8.2 and 1.4 Hz), 6.68 (1H, dd, J ¼ 8.2 from 12 (21 mg, 0.064 mmol) with 1-bromohexane to give 20.5mg of product

and 1.5 Hz), 6.64 (1H, d, J ¼ 1.5 Hz), 6.49 (1H, d, J ¼ 1.3 Hz), 3.90 (3H, s),

(94%). ¹H NMR (CDCl₃, 400 MHz): d ¼ 6.92ꢀ6.86 (3H, m), 6.75 (1H, d,

3.89 (3H, s), 3.81 (1H, d, J ¼ 13.0Hz), 3.54 (1H, d, J ¼ 13.0 Hz), 2.73 (1H, t, J ¼ 8.2 Hz), 6.51 (1H, s), 6.33 (1H, s), 4.02 (2H, t, J ¼ 6.8 Hz), 3.90 (3H, s),

J ¼ 4.2 Hz), 2.69 (1H, t, J ¼ 4.2 Hz), 2.66ꢀ2.58 (1H, m), 2.42ꢀ2.34 (1H, m),

2.86 ꢀ2.83 (2H, m), 2.60 ꢀ2.58 (2H, m), 2.51 ꢀ2.48 (2H, m), 2.19 (2H, t,

2.13 (1H, t, J ¼ 9.8 Hz), 1.81 ꢀ1.72 (1H, m), 1.54ꢀ1.37 (4H, m), 1.25ꢀ1.09 J ¼ 7.5 Hz), 1.80ꢀ1.73 (2H, m), 1.58 ꢀ1.51 (2H, m), 1.43ꢀ1.36 (2H, m),

(2H, m) and 0.94 ꢀ0.83 (1H, m)p.p.m. ¹³C NMR (CDCl₃, 100 MHz):

1.33 ꢀ1.27 (6H, m) and 0.85 (3H, t, J ¼ 6.6 Hz)p.p.m. ¹³C NMR (CDCl₃,

d ¼ 149.3, 148.7, 147.5, 147.2, 133.8, 133.6, 128.4, 126.9, 124.6, 124.0, 118.8, 100 MHz): d ¼ 211.1, 149.3, 148.4, 147.6, 146.9, 133.9, 133.6, 124.7, 122.8,

117.7, 112.5 (2C), 56.2 (2C), 53.0, 51.1, 34.0 (2C), 33.2, 30.1, 28.0 and 119.2, 118.7, 115.3, 113.0, 69.7, 56.3, 43.7, 41.6, 33.0, 31.6, 29.3, 29.2, 27.8,

23.7p.p.m. ESI-MS: calc. for C₂₈H₃₄NO₃[Mþ H]^þ: 432.3; found: 432.3. 25.6, 22.6, 21.3 and 14.0 p.p.m. ESI-MS: calc. for C₂₆H₃₅O₄[Mþ H]^þ: 411.3;

HPLC purity: 96.0% (254 nm), t_R: 5.92 min; 96.4% (220nm), t_R: 5.92min.

Compound 7d: 7d was prepared as described above for compound 7a from

4a (15.1 mg, 0.044 mmol) with hexylamine to give 14mg of product (74%). ¹H

found: 411.3. HPLC purity: 96.5% (254nm), t_R: 7.93min; 99.0% (220nm),

t_R: 7.93 min.

Compound 13d: 13d was prepared as described above for compound 13a

NMR (CDCl₃, 400 MHz): d ¼ 6.98 (1H, dd, J ¼ 8.2 and 1.2 Hz), 6.87 (1H, d, from 12 (20 mg, 0.06mmol) with 1-bromooctane to give 25 mg of product

J ¼ 8.2 Hz), 6.86 (1H, d, J ¼ 8.2 Hz), 6.75 (1H, dd, J ¼ 8.2 and 1.2 Hz), 6.61 (93%). ¹H NMR (CDCl₃, 400 MHz): d ¼ 6.93ꢀ6.86 (3H, m), 6.75 (1H, d,

(1H, s), 6.50 (1H, s), 3.89 (3H, s), 3.87 (3H, s), 2.94ꢀ2.59 (5H, m), 2.46ꢀ2.36 J ¼ 8.2 Hz), 6.51 (1H, s), 6.34 (1H, s), 4.02 (2H, t, J ¼ 6.8 Hz), 3.90 (3H, s),

(2H, m), 1.82ꢀ1.74 (2H, m), 1.67ꢀ1.43 (6H, m), 1.33 ꢀ1.25 (6H, m),

1.21ꢀ1.13 (1H, m) and 0.94ꢀ0.85 (4H, m)p.p.m. ¹³C NMR (CDCl₃,

2.86 ꢀ2.83 (2H, m), 2.61 ꢀ2.58 (2H, m), 2.51 ꢀ2.48 (2H, m), 2.20 (2H, t,

J ¼ 7.5 Hz), 1.81ꢀ1.73 (2H, m), 1.58 ꢀ1.51 (2H, m), 1.43ꢀ1.36 (2H, m),

100 MHz): d ¼ 149.7, 148.9, 147.6, 147.5, 133.4, 132.4, 125.1, 124.0, 118.8, 1.33 ꢀ1.23 (10H, m) and 0.86 (3H, t, J ¼ 6.6 Hz) p.p.m. ¹³C NMR (CDCl₃,

118.0, 112.7, 112.6, 56.2, 56.1, 54.8, 45.9, 33.1, 32.3, 32.1, 31.4, 29.8, 28.0, 27.4, 100 MHz): d ¼ 211.1, 149.3, 148.4, 147.6, 146.9, 133.9, 133.5, 124.7, 122.8,

26.7, 23.5, 22.5 and 14.0 p.p.m. ESI-MS: calc. for C₂₇H₄₀NO₃[Mþ H]^þ

119.2, 118.7, 115.3, 113.0, 69.7, 56.3, 43.7, 41.6, 33.0, 31.8, 29.4, 29.3, 29.2,

27.8, 25.9, 22.7, 21.3 and 14.1p.p.m. ESI-MS: calc. for C₂₈H₃₉O₄[Mþ H]^þ

426.3; found: 426.4. HPLC purity: 99.5% (254 nm), t_R: 6.15 min; 99.4%

(220nm), t_R: 6.15min.

Compound 7e: 7e was prepared as described above for compound 7a from

439.3; found: 439.3. HPLC purity: 97.3% (254nm), t_R: 8.25min; 98.5%

(220 nm), t_R: 8.25 min.

4a (15.2 mg, 0.045 mmol) with cyclohexylamine to give 14.7mg of product Compound 13e: 2-Bromopropane (45ml, 0.48 mmol) and N,N-diisopropy-

(78%). ¹H NMR (CDCl₃, 400 MHz): d ¼ 6.98 (1H, d, J ¼ 8.1 Hz), 6.89 (1H, d, lethylamine (DIPEA, 25ml, 0.14mmol) were added to the solution of 12

J ¼ 8.4 Hz), 6.88 (1H, d, J ¼ 8.3Hz), 6.77 (1H, dd, J ¼ 8.1 and 1.2 Hz), 6.61 (15.7mg, 0.048 mmol) in acetonitrile (10 ml). The reaction mixture was heated

(1H, s), 6.52 (1H, d, J ¼ 1.2 Hz), 3.90 (3H, s), 3.89 (3H, s), 3.05ꢀ3.01 (1H,

under reﬂux overnight. After the reaction was complete (monitored by HPLC),

m), 2.76ꢀ2.66 (3H, m), 2.53ꢀ2.42 (2H, m), 2.10 ꢀ2.07 (1H, m), 1.88ꢀ1.84 the solvent was removed by evaporation. The residue was dissolved in EtOAc

(2H, m), 1.79ꢀ1.62 (6H, m), 1.53ꢀ1.43 (3H, m), 1.26 ꢀ1.17 (5H, m) and (10 ml) and washed with water. The organic layer was dried over anhydrous

0.94ꢀ0.84 (1H, m)p.p.m. ¹³C NMR (CDCl₃, 100 MHz): d ¼ 149.7, 148.9,

Na₂SO₄and then ﬁltered. The ﬁltrate was concentrated in vacuo to yield the

147.6, 147.5, 133.4, 132.3, 125.3, 124.0, 118.8, 118.3, 112.6 (2C), 56.2, 56.1, crude residue, which was further puriﬁed by ﬂash column chromatography on

55.4, 52.5, 33.2, 32.8, 32.7, 32.3, 32.2, 29.8, 28.2, 25.1, 25.0 (2C) and 23.7p.p.m. silica gel (hexanes/ethyl acetate ¼ 91/9) to give 16mg of product (90%). ¹H

ESI-MS: calc. for C₂₇H₃₈NO₃[Mþ H]^þ: 424.3; found: 424.3. HPLC purity:

100% (254nm), t_R: 5.98min; 99.8% (220nm), t_R: 5.99min.

Compound 13a: The mixture of ketophenol 12 (18.6 mg, 0.057 mmol),

NMR (CDCl₃, 400 MHz): d ¼ 6.93 (1H, d, J ¼ 8.2 Hz), 6.89 (1H, d, J ¼ 8.2 Hz),

6.86 (1H, d, J ¼ 8.3 Hz), 6.75 (1H, d, J ¼ 8.2 Hz), 6.50 (1H, s), 6.33 (1H, d,

J ¼ 2.1 Hz), 4.56ꢀ4.46 (1H, m), 3.90 (3H, s), 2.86ꢀ2.83 (2H, m), 2.60 ꢀ2.58

benzyl bromide (9.5ml, 0.08mmol), and potassium carbonate (15.7 mg, (2H, m), 2.51 ꢀ2.48 (2H, m), 2.19 (2H, t, J ¼ 7.5Hz), 1.58ꢀ1.51 (2H, m) and

0.114 mmol) in acetone (5ml) was reﬂuxed for 6 h. The reaction mixture 1.33 ꢀ1.24 (8H, m)p.p.m. ¹³C NMR (CDCl₃, 100MHz): d ¼ 211.1, 148.5,

was ﬁltered and the ﬁltrate was evaporated under reduced pressure. Dichloro- 148.2, 148.1, 147.6, 134.7, 133.7, 124.7, 122.8, 119.3, 118.8 (2C), 113.2, 72.7,

methane (20 ml) was added to the residue, and the solution was washed with 56.4, 43.7, 41.6, 33.0, 29.3, 27.8, 22.3 and 21.3p.p.m. ESI-MS: calc. for

water (2ꢂ 30 ml), the organic layer was ﬁltered using a Biotage ISOLUTE C₂₃H₂₉O₄[Mþ H]^þ: 369.2; found: 369.2. HPLC purity: 97.8% (254nm), t_R:

phase separator (Biotage), evaporated and then puriﬁed by ﬂash column 7.34 min; 99.0% (220nm), t_R: 7.34min.

chromatography on silica gel (hexanes/ethyl acetate¼ 90/10) to give the

Compound 13f: At 0 1C, NaH (60% in mineral oil, 4.4 mg, 0.11mmol) was

added to a solution of 12 (18 mg, 0.055mmol) in DMF (2ml). To the resultant

product as a white powder (17.5mg; yield: 74%). ¹H NMR (CDCl₃,

400 MHz): d ¼ 7.31ꢀ7.30 (2H, m), 7.28 ꢀ7.27 (3H, m), 6.92 (1H, d, J ¼ 8.0 suspension, 3,3-dimethylallyl bromide (9.5ml, 0.083 mmol) was added at 0 1C.

Hz), 6.91 (1H, d, J ¼ 8.4 Hz), 6.83 (1H, dd, J ¼ 8.2 and 2.2 Hz), 6.78 (1H, dd, The mixture was then warmed up to room temperature and stirred for 2 h. The

J ¼ 8.2 and 2.0 Hz), 6.53 (1H, d, J ¼ 2.0 Hz), 6.34 (1H, d, J ¼ 2.0 Hz), 5.18 (2H,

s), 3.91 (3H, s), 2.85 ꢀ2.82 (2H, m), 2.60ꢀ2.57 (2H, m), 2.52ꢀ2.49 (2H, m),

2.19 (2H, t, J ¼ 7.4 Hz), 1.56ꢀ1.53 (2H, m) and 1.32ꢀ1.29 (2H, m)p.p.m.

reaction mixture was poured into water, and extracted with ethyl acetate.

The organic layer was dried over anhydrous Na₂SO₄, ﬁltered and evaporated.

The crude product was puriﬁed by ﬂash column chromatography on silica gel

¹³C NMR (CDCl₃, 100MHz): d ¼ 211.0, 148.7, 148.5, 147.4, 147.3, 137.4, (hexanes/ethyl acetate¼ 91/9) to give 12.6mg of product (58%). ¹H NMR

134.6, 133.6, 128.4, 127.7, 127.4, 124.6, 123.0, 119.1, 118.9, 116.5, 113.1, 56.3, (CDCl₃, 400 MHz): d ¼ 6.92 (1H, d, J ¼ 8.3 Hz), 6.90 ꢀ6.85 (2H, m), 6.76 (1H,

43.7, 41.6, 33.0, 29.2, 27.8 and 21.3p.p.m. ESI-MS: calc. for C₂₇H₂₉O₄[Mþ

H]^þ: 417.2; found: 417.2. HPLC purity: 99.6% (254 nm), t_R: 7.52 min; 99.5%

(220nm), t_R: 7.52min.

d, J ¼ 8.2 Hz), 6.50 (1H, s), 6.37 (1H, s), 5.48 (1H, t, J ¼ 6.5Hz), 4.60 (2H, d,

J ¼ 6.6 Hz), 3.89 (3H, s), 2.86ꢀ2.83 (2H, m), 2.61ꢀ2.58 (2H, m), 2.51 ꢀ2.48

(2H, m), 2.20 (2H, t, J ¼ 7.5 Hz), 1.73 (3H, s), 1.69 (3H, s), 1.58ꢀ1.52

Compound 13b: 13b was prepared as described above for compound 13a (2H, m) and 1.34ꢀ1.25 (2H, m) p.p.m. ¹³C NMR (CDCl₃, 100 MHz):

from 12 (21 mg, 0.064 mmol) with geranyl bromide to give 13mg of product d ¼ 211.1, 148.9, 148.5, 147.3, 147.2, 137.1, 134.0, 133.5, 124.5, 122.9, 120.4,

The Journal of Antibiotics

Novel diarylheptanoid analogs as antibacterial agents

L Shen et al

325

119.0, 118.8, 115.7, 112.9, 66.5, 56.2, 43.7, 41.5, 33.1, 29.2, 27.8, 25.8, 21.4 and

18.2 p.p.m. ESI-MS: calc. for C₂₅H₃₁O₄[Mþ H]^þ: 395.2; found: 395.2. HPLC

purity: 96.8% (254 nm), t_R: 7.56min; 98.5% (220nm), t_R: 7.56min.

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Biological studies

MIC determination. MIC values were determined against M. tuberculosis

(H37Rv) and other bacteria using the standard microbroth dilution method

exactly as described previously,²⁸which is based on the methods by the Clinical

and Laboratory Standards Institute.²⁹The maximum test concentration used

against M. tuberculosis and other pathogens was 200 mg ml^ꢀ1

Macromolecular synthesis. The effects of selected active compounds 7b–d (at

1 and 8 ꢂ MIC) on DNA, RNA and protein, respectively, were determined on

mid-logarithmic E. coli DTolC (OD₆₀₀¼ 0.4), by measuring the incorporation

of the radiolabeled precursors (methyl-³H)thymidine, (5,6-³H)uridine and

(4,5-³H)leucine into macromolecular fractions as described previously.^30,31

Brieﬂy, cells were mixed with tested compound for 5 min before the

addition of precursors at 1 mCiml^ꢀ1. After an additional 20min, cells were

lysed with cold 10% TCA and precipitated macromolecules collected on GF/C

ﬁlters. After washing with 95% ethanol, GF/C ﬁlters were analyzed by liquid

scintillation counting. The relative % activity in cells exposed to compounds

was determined. Ciproﬂoxacin (DNA), rifampicin (RNA) and streptomycin

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ACKNOWLEDGEMENTS

We thank the NIH Academic Research Enhancement Award (AREA)

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